Progress in the circadian rhythm field suggests that many key physiological mechanisms are remarkably dependent on the biological clock. It has also become clear that a number of diseases with important unmet medical need display marked circadian variation in their symptoms and severity. Indeed, abnormal functioning of the clock results in severe dysfunctions and pathologies, including cancer. As an example, the circadian organization tends to be lost and possibly replaced with an ultradian periodicity in rapidly growing or advanced stage tumors (Mormont et al., Int. J. Cancer, 1997; 70: 241-247).
Recent developments in our understanding of circadian biology also indicate that time and duration of dosing may have profound consequences for the efficacy and safety of new and existing therapeutic agents. Indeed, an increasing number of drugs show a circadian-dependency of their pharmacodynamics (Paschos et al., Annu. Rev. Pharmacol. Toxicol., 2010; 50: 187-214).
Circadian dosing time appears to influence the extent of toxicity of numerous anticancer drugs, including cytostatics and cytokines, in mice or rats (Paschos et al., Annu. Rev. Pharmacol. Toxicol., 2010; 50: 187-214). For all these drugs, the survival rates are reported to vary by 50% according to a circadian dosing time of a potentially lethal dose. Such a large difference is observed irrespective of injection route—(intravenous or intraperitoneal) or the number of injections (single or repeated)—(Mormont et al., Cancer, 2003; 97: 155-169).
A remarkable observation reveals that risk for breast cancer is significantly higher in industrialized societies, and the risk increases as developing countries become more westernized (Sahar et al., Cell Cycle, 2007; 6: 1329-1331). Incidence of breast cancer increases significantly in women working nightshifts, being higher among individuals who spent more years and hours per week working at night. From a clinical point of view, prognosis of cancer is poorer in patients with altered circadian rhythm compared to patients with normal rhythm (Sahar et al., Cell Cycle, 2007; 6: 1329-1331).
In 2007, the International Agency for Research on Cancer (IARC) classified shift work with circadian disruption or chronodisruption as a probable human carcinogen (Erren et al., Dtsch. Arztebl. Int., 2010; 107: 657-662). Interestingly, disruption of circadian endocrine rhythms, either by pinealectomy or through constant light exposure, increases the formation of spontaneous mammary tumors in rodents (Sahar et al., Cell Cycle, 2007; 6: 1329-1331).
Breast cancer is the second most common type of cancer after lung cancer worldwide and the fifth most common cause of cancer death. It is found more in women as compared to men. Worldwide more than one million new cases of breast cancer are diagnosed every year. New breast cancer cases are mostly found in North America. Breast cancer is usually found in women over the age of 40 years. Breast cancer cases have risen about 30% in the past 25 years in western countries like the US and Europe.
Although a continuous perturbation of the circadian rhythm has been proposed as a probable human carcinogen, a surprising observation showed that some alteration of the circadian clock may include beneficial effects. As an example, in mice bearing a deletion of the tumor suppressor p53 the genetic ablation of the two circadian regulators CRY1 and CRY2 decreased the development of tumors and increased the survival of the animals (Ozturk et al., Proc. Natl. Acad. Sci. U.S.A, 2009; 106: 2841-2846).
Taken collectively, these observations open up the possibility to identify novel pharmacological targets for cancer treatment among the molecular factors involved in the circadian regulation.
A decade ago, the REV-ERB/NR1D subgroup receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), was identified as an integral component of the circadian clock machinery (Teboul et al., J. Appl. Physiol., 2009; 107: 1965-1971; Ripperger et al., Cell Res., 2012; 22:1319-1321). Both REV-ERBs receptors lack the classical nuclear receptor AF-2 trans-activating domain and represses gene activity by associating with a co-repressor complex (Ripperger et al., Cell Res., 2012; 22:1319-1321; Phelan et al., Nat. Struct. Mol. Biol., 2010; 17: 808-814).
In the presence of the natural ligand (heme), REV-ERB/co-repressor complex interaction is enhanced and the transcriptional repression of target genes is achieved (Ripperger et al., Cell Res., 2012; 22:1319-1321). The selection of the targets is obtained by the binding of the DNA binding domain of REV-ERBs with specific cis-elements within the promoter of the genes.
Being historically first discovered, the biological function of REV-ERBα has been the most characterized. Indeed, REV-ERBα has been implicated in the regulation of several processes, including circadian rhythm, adipogenesis, inflammation (Phelan et al., Nat. Struct. Mol. Biol., 2010; 17: 808-814).
At the molecular level, several genes, which activity is regulated by REV-ERBα, have been reported (Phelan et al., Nat. Struct. Mol. Biol., 2010; 17: 808-814). Notably, in addition to genes involved in the above mentioned processes, REV-ERBα regulated targets also include important cell cycle regulatory genes, such as the Cyclin-dependent kinase inhibitor p21 (Cink1a/p21) (Burke et al., Nucleic Acids Research, 1996; 24: 3481-3489; Borgs et al., Cell Cycle, 2009; 8: 832-837).
REV-ERBα has been also reported to repress the transcription of genes such as Elovl3 (a very long-chain fatty acid elongase) (Anzulovich et al., J. Lipid Res., 2006; 47: 2690-2700) and PAI-1 (Plasminogen Activator Inhibitor 1, a regulator of the fibrinolytic system and modulator of inflammation, atherothrombosis and atherosclerosis) (Wang et al., J. Biol. Chem., 2006; 281: 33842-33848). Because several responses mediated by REV-ERBα feel the influence of the circadian rhythm, it has been postulated that REV-ERBα activity may have the potential to contribute to—or even to control—the crosstalk between circadian and many other physiological processes.
However, due to the modest clock phenotypes of REV-ERBα-deficient mice, REV-ERBα has been proposed to confer robustness to the oscillatory clock rather than being a pacemaker component essential for rhythm generation, such as other circadian regulators (i.e., Bmal1) (Teboul et al., J. Appl. Physiol., 2009; 107: 1965-1971). This view has been recently challenged by a recent publication that strongly supports a much more prominent role of REV-ERBs receptors in the core clock mechanism than anticipated (Cho et al., Nature, 2012; 485: 123-127). In fact, the comparative analysis of genome sequences on which REV-ERBα and REV-ERBβ are recruited revealed an extensive overlap between the cistromes of the two receptors (Cho et al., Nature, 2012; 485: 123-127). This result suggests that REV-ERBα and REV-ERBβ isoforms can compensate each other for the repression of several target genes, including several members of the circadian regulators.
In line with this observation, the generation of liver-specific REV-ERBα/REV-ERBβ double-knockout mice (L-DKO) demonstrated that 90% of the approximately 900 genes rhythmically expressed in the liver of wild-type animals became arrhythmic in the liver of the L-DKO mice (Cho et al., Nature, 2012; 485: 123-127).
Nevertheless, some biological responses may depend on the activity of a specific REV-ERBs isoform. Indeed, genetic ablation of REV-ERBα or genetic knockdown of REV-ERBα expression have been reported to modulate the production and release of the proinflammatory cytokine IL-6 (Gibbs et al., Proc. Natl. Acad. Sci. U.S.A, 2012; 109: 582-587).
A role of REV-ERBα as a survival factor for breast cancer cells with the ErbB2 signature has been also reported (Kourtidis et al., Cancer Research, 2010; 70: 1783-1792). The proto-oncogene ErbB2 (Her2) is overexpressed in about the 30% of breast carcinoma (ErbB2 positive breast tumors). REV-ERBα has been reported to be co-overexpressed with ErbB2, suggesting that this gene may represent novel factor influencing ErbB2-positive tumors. More importantly, the genetic inhibition of REV-ERBα was able to block the proliferation of ErbB2 positive breast cancer cells (Kourtidis et al., Cancer Research, 2010; 70: 1783-1792).
Notably, it has been recently reported that the protein DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of REV-ERBα (Chini et al., The Biochemical Journal, 2013; 451: 453-461). DBC1 was originally identified during a genetic search for candidate breast tumor suppressor genes on a human chromosome 8p21 region frequently deleted in breast cancers (Trauernicht et al., Cell Cycle, 2010; 9: 1218-1219). However, further analyses revealed that DBC1 expression is not substantially lost in cancers from any source. In fact, DBC1 has been found to be upregulated in breast carcinoma versus normal breast tissue and in breast ductal carcinoma versus other cancers (Trauernicht et al., Cell Cycle, 2010; 9: 1218-1219). The fact that DBC1 enhances REV-ERBα protein stability (Chini et al., The Biochemical Journal, 2013; 451: 453-461) reveals an interesting molecular connection between DBC1 and REV-ERBα overexpressing breast tumors.
Although the molecular mechanism of REV-ERBα mediated growth inhibition of ErbB2-positive breast cancer cells has not deeply been investigated yet, biological data (Kourtidis et al., Cancer Research, 2010; 70: 1783-1792) support the idea that a pharmacological repression of REV-ERBα activity may be used for breast cancer therapy.
In addition, considering that REV-ERBα and REV-ERBβ showed a co-recruitment on the promoters of several genes involved in cell cycle regulation, including p21 (Borgs et al., Cell Cycle, 2009; 8: 832-837; Cho et al., Nature, 2012; 485: 123-127), the co-repression of both REV-ERBs isoforms may even have more pronounced effects on the proliferation and viability of cancer cells.
The first REV-ERBα ligand, the agonist tertiary amine GSK4112, has been recently identified (Grant et al., ACS Chemical Biology, 2010; 5: 925-932). GSK4112 was identified in a FRET assay as capable to dose-dependently increase the interaction of a peptide derived from NCoR (Nuclear receptor Co-Repressor) with REV-ERBα (Grant et al., ACS Chemical Biology, 2010; 5: 925-932). The treatment with GSK4112 decreases BrnaH expression in cell culture in a dose-dependent manner and induces adipogenesis in 3T3-L1 cells as demonstrated by lipid accumulation and increased expression of key adipogenic genes (Kojetin et al., Current Pharm. Design, 2011; 17: 320-324). GSK4112 behaves therefore as a REV-ERBα agonist, regulating the expression of REV-ERBα responsive target genes in a manner similar to the physiological ligand, heme (Ripperger et al., Cell Res., 2012; 22:1319-1321).
The ligand binding domain (LBD) of REV-ERB shares a high homology degree with the REV-ERBβ LBD. Indeed, GSK4112 has been reported to act on both isoforms (Solt et al., Nature, 2012; 485: 62-68). GSK4112 displays poor pharmacokinetic properties because of high clearance and rapid metabolism that decrease its bioavailability. (Solt et al., Nature, 2012; 485: 62-68).
REV-ERB ligands, such as the REV-ERB agonists SR9009, SR9011 and analogs, are known from WO 2013/033310. They are closely related to GSK4112 by sharing a common tertiary amine scaffold and two out of the three substituents at the amine nitrogen, and they are disclosed as useful treatments of malconditions such as diabetes, obesity, atherosclerosis, dyslipidemia and coronary artery disease.
Compounds SR9009 and SR9011 have been tested in mice for their possibility to induce metabolic responses (Solt et al., Nature, 2012; 485: 62-68). These compounds, in addition to influence the circadian clock, were also able to improve the metabolic parameters of diet-induced obese mice. The changes measured on biochemical parameters were also associated to a modified gene expression profile in metabolic tissues such as liver, skeletal muscle and adipose tissue. However, the use of SR9009 and SR9011 to interrogate REV-ERBα biology is complicated by high metabolic clearance rates that necessitate high dosing to achieve meaningful levels of exposure in vivo (Solt et al., Nature, 2012; 485: 62-68). Furthermore, the unfavorable DMPK (Drug Metabolism and Pharmacokinetic) profile of GSK4112, SR9009 and SR9011 renders these compounds unsuitable as truly effective remedies for the clinical treatment of diseases and pathologies associated to the unbalance of the circadian clock.
In addition, the tertiary amines mentioned above have known activity on the nuclear receptor LXRα, a potential liability for interpretation of results from cell-based and animal pharmacology (Trump et al., J. Med. Chem., 2013; 56: 4729-4737). Indeed, further studies demonstrated that GSK4112 also agonizes LXRα. Notably, SR9009 and SR9011 resulted 100 times more active toward LXRα than REV-ERBs (Trump et al., J. Med. Chem., 2013; 56: 4729-4737). Novel triarylmethylamines related to GSK4112, SR9009 and SR9011 and showing high REV-ERBα agonist potency, selectivity, and bioavailability have been reported (Trump et al., J. Med. Chem., 2013; 56: 4729-4737). Notably, these GSK4112 analogues displayed a much lower effect than GSK4112 on the reduction of LPS-mediated IL-6 transcriptional activation.
WO 2013/045519 claims certain substituted triazolopyridazines as REV-ERB agonists useful for treating any disease wherein the activation of REV-ERB has therapeutic effects, for instance in inflammatory and circadian rhythm-related disorders or cardiometabolic diseases.
Finally, certain dibenzylamine derivatives are claimed in WO 2007/065261 as inhibitors of PAI-1 and, as such, claimed to be therapeutically useful, mainly, in several cardiovascular diseases and noninsulin dependent diabetes mellitus.
The only reported REV-ERBα antagonist described in the literature is the 1,2,3,4 tetrahydroisoquinoline derivative SR8278 which, differently from GSK4112, features an amidic nitrogen as the core element, thus shifting away from basic compounds (Kojetin et al., ACS Chem. Biol., 2011; 6: 131-134). SR8278 is described as a REV-ERBα antagonist based on its ability to inhibit REV-ERBα LBD/NcoR interaction in a FRET-based assay and to induce the expression of two REV-ERBα target genes in cultured cells (Kojetin et al., ACS Chem. Biol., 2011; 6: 131-134). These results suggest that SR8278 acts with a molecular mechanism similar to GSK4112, i.e. REV-ERB/NcoR association, but with an opposite outcome. Also SR8278, as mentioned by its disclosers, displayed poor pharmacokinetic properties limiting its pharmacological uses (Kojetin et al., ACS Chem. Biol., 2011; 6: 131-134).
There is thus the need in the art to find new REV-ERB antagonists.